title: Restoration of anti-Aspergillus defense by neutrophil extracellular traps in human chronic granulomatous disease after gene therapy is calprotectin-dependent.
abstract: BACKGROUND
  Aspergillus spp infection is a potentially lethal disease in patients with neutropenia or impaired neutrophil function. We showed previously that Aspergillus hyphae, too large for neutrophil phagocytosis, are inhibited by reactive oxygen species-dependent neutrophil extracellular trap (NET) formation. This process is defective in chronic granulomatous disease (CGD) because of impaired phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase function.
  
  
  OBJECTIVE
  To determine the antifungal agent and mechanism responsible for reconstitution of Aspergillus growth inhibition within NETs after complementation of NADPH oxidase function by gene therapy (GT) for CGD.
  
  
  METHODS
  Antifungal activity of free and NET-released calprotectin was assessed by incubation of Aspergillus nidulans with purified calprotectin, induced NETs from human controls, and CGD neutrophils after GT in the presence or absence of Zn(2+) or α-S100A9 antibody, and with induced NETs from wild-type or S100A9(-/-) mouse neutrophils.
  
  
  RESULTS
  We identified the host Zn(2+) chelator calprotectin as a neutrophil-associated antifungal agent expressed within NETs, reversibly preventing A nidulans growth at low concentrations, and leading to irreversible fungal starvation at higher concentrations. Specific antibody-blocking and Zn(2+) addition abolished calprotectin-mediated inhibition of A nidulans proliferation in vitro. The role of calprotectin in anti-Aspergillus defense was confirmed in calprotectin knockout mice.
  
  
  CONCLUSIONS
  Reconstituted NET formation by GT for human CGD was associated with rapid cure of pre-existing therapy-refractory invasive pulmonary aspergillosis in vivo, underlining the role of functional NADPH oxidase in NET formation and calprotectin release for antifungal activity. These results demonstrate the critical role of calprotectin in human innate immune defense against Aspergillus infection.
authors.affiliation: Division of Immunology/Hematology/BMT, University Children's Hospital Zurich, Zurich, Switzerland.
authors.forename: Matteo
authors.initials: M
authors.lastname: Bianchi
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Niemiec
authors.forename: Ulrich
authors.initials: U
authors.lastname: Siler
authors.forename: Constantin F
authors.initials: CF
authors.lastname: Urban
authors.forename: Janine
authors.initials: J
authors.lastname: Reichenbach
mesh_terms: D000818:Animals; D000935:Antifungal Agents / Q000378:metabolism / Q000494:pharmacology; D001228:Aspergillosis / Q000276:immunology* / Q000382:microbiology; D001233:Aspergillus nidulans / Q000187:drug effects*; D002648:Child; D015316:Genetic Therapy / Q000379:methods*; D006105:Granulomatous Disease, Chronic / Q000150:complications / Q000276:immunology / Q000628:therapy*; D006801:Humans; D039841:Leukocyte L1 Antigen Complex / Q000378:metabolism* / Q000494:pharmacology; D008297:Male; D051379:Mice; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D009504:Neutrophils / Q000276:immunology* / Q000378:metabolism / Q000502:physiology; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D000935:Antifungal Agents; D039841:Leukocyte L1 Antigen Complex; D017382:Reactive Oxygen Species; D019255:NADPH Oxidases